The about 80kD erythroid membrane skeletal component, protein 4.1, is the prototypical member of a complex family of structural protein isoforms encoded by a single genetic locus via multiple alternative pre- mRNA splicing pathways. Whereas the structure of the major red cell 4.1 isoforms has been defined, and some of its functional domains well characterized, a molecular genetic explanation for the diversity in 4.1 size (30-210kD) and intracellular localization (peripheral membranes, stress fibers, nuclei, centrosomes, Golgi) is lacking. In order to completely define the genetic repertoire of the 4.1 gene, and to characterize developmental switched in 4.1 expression mediated by regulated transcription and splicing events, the following aims are proposed. (1) Determine the DNA sequence of the entire about 250kb human 4.1 gene locus, to provide a wealth of primary data essential for analysis of gene function in normal individuals and in selected patients with hereditary elliptocytosis. (2) Explore the structural determinants of 4.1 isoform localization, by (a) characterizing transcription- and splicing-mediated changes in the complement of 4.1 mRNAs expressed in differentiating erythroid and epithelial cells, and (b) determining the subcellular compartmentalization of individual epitope-tagged isoforms of known primary sequence. (3) Develop genetic approaches toward manipulation of 4.1 expression in tissue-specific and isoform-specific fashion, by generating mice with 4.1 gene disruptions and rearrangements. 4.1 gene knockouts by homologous recombination in ES cells, and gene rescue experiments with normal or mutated YAC transgenes, are proposed to explore the biological significance of selected isoforms or functional domains. Studies will focus primarily on erythroid and epithelial cells, in which regulated switches in 4.1 expression have been demonstrated, and on fibroblasts, in which nuclear 4.1 isoforms have been identifies. The protein 4.1 gene will serve as a valuable model for exploring tissue-specific expression of a large and highly complex gene encoding a critical component of multiple intracellular skeletal structures. Successful accomplishment of these aims will facilitate elucidation of the biological functional significance of this diverse protein family, and may provide insight into the pathophysiologic consequences of disruptions in 4.1 gene expression.